Tremendous advances in ion channel research have been made with respect to channel structure and function. However, there is a lack of information regarding how living cells process ion channel proteins in real time. Such information is important since rapid modulation of ion channel surface expression is likely to represent a central mechanism in the regulation of cellular electrical excitability. Human diseases have already been linked to ion channel localization and trafficking defects. The Kv2.1 delayed-rectifier K+ channel targets to unique cell surface clusters in hippocampal neurons. Since these structures are regulated in vivo by stimuli associated with neuronal injury, i.e. hypoxia, ischemia, and excess neurotransmitter release, these microdomains are likely to participate in the neuro-protective response during such insults. However, Kv2.1 also plays a role in apoptosis, with this Kv isoform providing the increased K+ permeability required for the completion of the apoptotic program in cortical neurons. These diverse functions mandate that Kv2.1 activity and trafficking be tightly regulated. Our overall hypothesis is that the Kv2.1 surface clusters are central in the regulation of both Kv2.1 trafficking and function. Thus, a better understanding of cluster function will improve our ability to manage stroke-related issues. By clustering these channels within a defined surface compartment that serves as a platform for both insertion and retrieval, the regulation of trafficking is more efficient than if the channel is homogenously distributed over the cell surface. In addition, our preliminary data suggest that channels within the surface clusters do not conduct K+ until released from these cell surface domains, suggesting that K+ current density is controlled by more than transport to the cell surface. The proposed research will employ a multidisciplinary approach utilizing live cell imaging, single channel detection/tracking, site-directed mutagenesis and electrophysiology. The three Specific Aims will test the hypotheses that 1) Kv2.1 clusters represent cell surface platforms for channel insertion and retrieval, 2) stimulus-induced modification of the cortical cytoskeleton causes immediate release from the cell surface cluster and 3) Kv2.1 surface clusters are cell surface storage sites containing non-conducting channel. This research will have implications far beyond neurobiology, for Kv2.1 is the most widely expressed Kv channel and is also physiologically important to the cardiovascular and endocrine systems. PUBLIC HEALTH RELEVANCE: The proposed research examines the localization of the Kv2.1 potassium channel in brain neurons. This localization is essential in the neuro-protective response to stroke injury and hyperactivity in the brain. Mice lacking the Kv2.1 channel are epileptic and hyper-excitable. Understanding the molecular mechanisms underlying Kv2.1 localization will enhance the treatment of stroke and epilepsy.